Biocompatibility of Mineral Trioxide Aggregate Mixed with Different Accelerators: an Animal Study

Statement of the Problem: Several additives have been introduced to decrease the setting time of MTA (mineral trioxide aggregate). For clinical applications, it is essential to investigate the biocompatibility of these materials. Purpose: The present study evaluated the tissue response to MTA that has been separately mixed with citric acid, calcium lactate gluconate (CLG), and Na2HPO4. Materials and Method: In this experimental study Twenty one Wistar rats were divided into three groups of 7, 14 and 30 days follow up periods. Sterile polyethylene tubes were subsequently filled with MTA separately mixed with distilled water, 0.1% citric acid, 0.43% calcium lactate gluconate (CLG) and 15% Na2HPO4 and afterwards implanted subcutaneously. Empty tubes were implanted as negative control. At the end of their respective periods, the animals were sacrificed by anesthetic overdose and a biopsy was performed. The inflammatory responses were scored, classified and statistically analyzed using Kruskal-Wallis and Man-Whitney tests. Statistical significance was defined as p< 0.05. Results: There was no significant difference between test groups in any time period after implantation but the mean values of inflammatory responses were significantly more than that of the negative control group (p> 0.05). The mean values of inflammatory responses were decreasing over time in all test groups. These values did not significantly differ in any group except the CLG and Na2HPO4 groups. Conclusion: The inflammatory responses induced by MTA mixed with citric acid and MTA mixed with Na2HPO4 were comparable to that of the control MTA. MTA mixed with CLG provoked a moderate-to-severe inflammatory response at 7 days after implantation, so further study is required before clinical application of this cement.


Introduction
When the pulp is exposed by dental caries or trauma, vital pulp therapy becomes the procedure of choice to preserve the primary teeth until their natural shedding [1][2]. After removing the infected and inflamed coronal pulp, the vital, uninfected radicular pulp tissue is covered by formocresol [3], ferric sulfate [4] or MTA (mineral trioxide aggregate) [3].
There is no consensus about the best amongst the various published methods of pulpotomy [3,5].
Formocresol has been the most common pulp capping material used in the last six decades. In spite of systemic absorption of formaldehyde (the major component of formocresol), pulpotomy with formocresol has shown success rate of 97%. The mutagenic and carcinogenic effects of formocresol on the pulp have been the subject of many studies over the past 20 years [6][7].
In recent years, the use of MTA for pulp-capping has been proposed as an alternative to formocresol [8].
MTA is a white or gray powder consisting of fine hydrophilic particles of tricalcium silicate, tricalcium oxide, tricalcium aluminate, and silicate oxide. MTA has been used in vital pulp therapies, in repairing furcal and lateral perforations and as a root-end filling material during apical surgery, because of its biological properties [9][10][11][12]. MTA shows antimicrobial and dentinogenic effects on the pulp and preserves pulp integrity after pulp-capping or pulpotomy. A systematic study about pulpotomy with MTA in primary teeth has shown that this method results in a lower failure rate, less internal resorption, and leads to greater success [13].
Despite the favorable properties of MTA, it has some disadvantages. The major disadvantages are its long setting time (75 minutes to 72 hours), difficult handling, and high cost [14][15]. Many studies have focused on these limitations and several accelerators have been introduced to decrease the long setting time of MTA cement [15][16][17][18][19][20]. It has been reported that mixing MTA with additives such as Na 2 HPO 4 [17], citric acid, and calcium lactate gluconate (CLG) [20] significantly decreases the setting time, although few studies have been conducted on the biocompatibility of these materials [15,19,[21][22]. For clinical applications, it is essential to investigate the biocompatibility of new materials. The present study evaluated the biocompatibility of MTA mixed with the three different accelerators mentioned above.  The rats were then divided into 3 groups, each consisting of 7 rats, and maintained for periods of 7, 14 and 30 days. At the end of their respective periods, the animals were dispatched by administering a high dose of anesthetic and a biopsy was performed on a 2.5 mm diameter tissue surrounding each test tube. Adhering to the hygienic principles, the animals were buried in a special place provided before for such projects. After fixation of the resected tissue samples in 10% formalin, they were serially sectioned into 4-ϻm-thick samples and stained with hematoxylin and eosin. The sections were evaluated by a pathologist using blind analysis with a light microscope (Olympus BX41, Japan) using 10× and 40× objective lenses. The inflammatory responses after implantation were scored and classified according to previously established scoring system [23] as (0) for no reaction; absence of inflammatory cells, (1+) for mild reaction; presence of mild chronic inflammatory infiltrate or <25 eosinophilic or giant cells, (2+) for moderate reaction; moderate chronic inflammatory infiltrates or 25-150 eosinophilic or giant cells, and (3+) for severe reaction, intense chronic inflammatory infiltrate or >150 eosinophilic or giant cells [23]. the histological differences between the test materials.

Materials and Method
The differences in inflammatory responses for the three time periods were examined using Kruskal-Wallis and Man-Whitney tests. Statistical significance was defined as p< 0.05.

Results
The mean and standard deviation of inflammatory responses in different periods are shown in Table 1. There was no significant difference between experimental groups in any time period after implantation but the mean values of inflammatory responses of all experimental groups were significantly more than negative control group. This amount of difference in the mean inflammatory values is shown in Table 1.
The mean values of inflammatory responses were decreasing over time in all test groups. These values were not statistically different in any group except groups 3 and 4. In group 3, the inflammatory response on the 7 th day after implantation was significantly more than that of the 30-days implantation (p= 0.01). In group 4, the inflammatory response seen at day 7 after implantation, was significantly more than that observed on 14 th and 30 th days (p= 0.02). Photomicrographs of different inflammatory reactions of different experimental groups are presented in figures 1 to 4 and the mean inflammatory score of the test groups are shown in Figure 5.

Discussion
According to the results of this study, adding 0.1% citric acid, 43.4% CLG and 15% Na 2 HPO 4 did not significantly affect the tissue response to MTA.     of any such effects of new materials to adjacent tissues.
One way to evaluate the biocompatibility of such materials is to implant them in subcutaneous tissue and observe the inflammatory responses [24].
Subcutaneous implantation of materials in small laboratory animals can simulate in situ conditions of the material [25]. Mutoh et al. [26] reported that implanting materials using sterile tubes prevents the release of the substance into adjacent tissue. These tubes resemble a root canal of the tooth and are more advantageous than placing the material directly into the tissue. The inert nature of polyethylene tubes makes them suitable for implantation studies. In the present study, sterile polyethylene tubes were used for implantation. In this study, just as shown in previous studies [27][28][29][30], no reaction or mild inflammatory response decreasing over time was seen after implanting the empty negative control tubes.
The initial inflammatory response to the empty tubes is considered a response to the surgical procedure of implantation [30].  [29][30][31][32]. The initial inflammatory response followed by MTA implantation can be explained by a response to pH, the heat generated during setting, and the production of inflammatory cytokines such as IL1 and IL6 in the beginning of the process [33].
In the present study, there were no significant differ- ed a "good" response. In their study, this material showed a higher rate of cell viability than control MTA and the MTA mixed with Na2HPO4 at day 7 of experimentation. Lee et al. [35] reported that low-dose citric acid had no adverse effects on biocompatibility, osteogenic differentiation, and mineralization of MTA.
CLG is a soluble salt of calcium, lactic acid and gluconic acid that is often used in effervescent calcium tablets. It is reported that this material can decrease the time-setting period of MTA to 13.9 minutes [20]. In the present study, no significant differences were seen between the histological response of this cement and the control MTA; however, analysis with more details showed an inflammatory reaction on the 7 th day that significantly differed from that of 30 th days. This cement showed moderate-to-severe inflammatory response (more than any other experimental groups) on day 7, which decreased to a mild reaction after 30 days (less than the others did).
Ji et al. [ [41][42][43]. Further studies should be conducted to clarify the effect of accelerators on mechanical properties of MTA.

Conclusion
For all of the accelerators tested in this study, the inflammatory response decreased over time. The results of this study indicate that the inflammatory responses induced by MTA mixed with 0.1% citric acid and MTA mixed with 15% Na2HPo4 were comparable to that of the MTA mixed with distilled water. These cements appear to be biocompatible within the limitations of this study. Despite the absence of significant differences between control MTA and MTA mixed with CLG for histological response, the latter cement provoked a moderate-to-severe inflammatory response on 7 th day after implantation. Further studies are suggested before clinical application of this cement.